Method of producing biologically active human acidic fibroblast growth factor and its use in promoting angiogenesis

ABSTRACT

The gene of human acidic fibroblast growth factor 155 (haFGF 155) has been obtained by chemical synthesis. The nucleotide sequence of haFGF 155 gene has been deduced on the basis of haFGF 155 amino acid sequence as described in the literature. The amino acid sequence, of the synthesized haFGF 155 does not differ from those described in the literature. The nucleotide sequence of haFGF gene differs from those described previously. For chemical synthesis of haFGF 155 gene, codons were used which are the ones most often used by  E. coli  in highly expressed  E. coli  proteins. A plasmid with haFGF 155 (phaFGF 155) gene was obtained and was used to transform  E. coli . Production of haFGF 154 protein was achieved by cultivation of the producer strain under conditions which slow down the lytic development of lambda phage. The haFGF 154 protein accumulated in culture medium in a soluble condition as a result of the producer strain cells lysis by the lambda phage. The haFGF 154 protein constituted 20% of the soluble protein accumulated in the culture medium and its biological activity was demonstrated by its ability to generate new vessels (angiogenesis). The initiator methionine residue at position 1 of the FGF 155 protein was completely removed during protein synthesis resulting in an FGF 154 amino acid product. The use of the phage-dependent method to produce other forms of the haFGF protein is also disclosed.

RELATED APPLICATIONS

[0001] The present application is a divisional application of U.S.application Ser. No. 09/925,945, filed Aug. 15, 2001 which claimspriority to provisional patent application serial No. 60/225,406,entitled, “A Method of Producing Biologically Active Human AcidicFibroblast Growth Factor and Its Use in Promoting Angiogenesis,” filedon Aug. 15, 2000.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The field of the invention relates to methods of producing arecombinant fibroblast growth factor protein and its use in promotingangiogenesis.

[0004] 2. Description of the Related Art

[0005] Fibroblast growth factors (FGF) are nine structurally relatedpolypeptides, which are potent regulators of cell proliferation,differentiation and normal development. They also take part inpathological processes of tumorogenesis and metastasis (Galzie, et al.Biochem. Cell Biol. (1997) 75: 669-685). They are potent mitogens anddifferentiation factors for a broad range of mesoderm and neuroectodermderived cells, including endothelial cells.

[0006] The heparin proteoglycans, heparin or heparin sulfate, bindseveral FGF molecules together as a complex which are presented to theFGF receptors. FGF proteins bind to their receptors resulting in theactivation of protein tyrosine kinases. The phosphorylation of thesetyrosine kinases initiates multiple signals including the transcriptionof new mRNA's.

[0007] Two fibroblast growth factors, basic and acidic, are described aspotent inducers of angiogenesis (Friesel et al. (1995) FASEB J. 9:919-925). Both basic and acidic factors have been implicated in thecontrol of blood vessel formation and their involvement in normal andpathological angiogenesis (Slavin, J. (1995) Cell Biology International19(5): 431-444). These factors have been purified, their amino acidsequences have been determined and their CDNA has been cloned andsequenced.

[0008] Acidic Fibroblast Growth Factor (aFGF) has been described undervarious names including embryonic kidney-derived angiogenesis factor I,astroglial growth factor I, endothelial cell growth factor (ECGF),retina-derived growth factor, heparin-binding growth factor class 1,endothelial growth factor, eye-derived growth factor II, prostatropin,and glial maturation factor (Gospodarowicz, et al. (1987) Journal ofCellular Physiology supplement 5: 15-26). Cloning, nucleotide sequenceand chromosome localization have been described (Jaye et al. (1986)Science 233: 541-545).

[0009] The aFGF gene is situated on chromosome 5. It has a single copyand encodes three exons separated by two introns. A 4.8 kb mRNAtranslates synthesis of a form of aFGF with 155 amino acids. However,the N-terminal methionine residue is removed in vivo to give a 154 aminoacid form. This 154 amino acid form of the aFGF is processed into twoforms which are 140 and 134 amino acids. The aFGF protein is an anionicmitogen of molecular weight 15,000-17,000 D.

[0010] The aFGF protein has been found in brain, retina, bone matrix andosteosarcoma. Only forms with 140 and 134 amino acids have been obtainedfrom tissues. It has been suggested that the truncated aFGF forms are anartifact created by specific proteases during aFGF extraction andisolation (Gospodarowicz, et al. (1987) Journal of Cellular Physiologysupplement 5: 15-26; Jaye et al.(1987) The Journal of BiologicalChemistry 262 (34):16612-16617).

[0011] It has been suggested that heparin potentiates the biologicalactivity of the aFGF protein (Thornton et al. (1983) Science 222 (4624):623-625). Heparin binding to aFGF has been observed (Maciag et al.(1984) Science 225 (4665): 932-935). This heparin-binding characteristichas been used as an efficient affinity chromatography method for thepurification of aFGF protein. Heparin potentiates the biologicalactivity of aFGF and the enhanced activity of the aFGF-heparin complexvaries from several to one hundred fold (Lobb, et al. (1986) Anal.Biochem. 154: 1-14).

SUMMARY OF THE INVENTION

[0012] In one embodiment, the present invention relates to a method forproducing a biologically active human acidic fibroblast growth factorprotein, including the steps of:

[0013] transforming a strain of E. coli with a plasmid having at leastone copy of an expressible gene encoding a biologically active humanacidic fibroblast growth factor protein, operably linked to a promoter;

[0014] infecting the transformed bacterial host cell with abacteriophage λ capable of mediating delayed lysis; and

[0015] cultivating the E. coli host cell under a culture condition thatinduces lytic growth of said cell without lysis until a desired level ofproduction of said protein is reached, wherein said protein is producedas a soluble, biologically-active human acidic fibroblast growth factorprotein.

[0016] In a preferred embodiment, the bacteriophage λ has atemperature-sensitive mutation. In a more preferred embodiment, thetemperature-sensitive mutation is cI₈₅₇.

[0017] In a preferred embodiment, the E. coli host cells are grown at atemperature which prevents lytic growth of the bacteriophage λ prior tothe cultivating step.

[0018] In a preferred embodiment, the bacteriophage λ has a mutation inat least one gene capable of mediating delayed lysis. In a morepreferred embodiment the at least one gene capable of mediating delayedlysis is selected from the group consisting of N, Q and R.

[0019] In a preferred embodiment, the strain of E. coli produces asuppressor for the repair of amber-mutations.

[0020] In an alternate embodiment, the strain of E. coli lacks asuppressor for the repair of amber-mutations.

[0021] In a preferred embodiment, the infecting bacteriophage λ isprovided at a multiplicity of infection in a range of about 1 to about100. In a more preferred embodiment, the infecting bacteriophage λ isprovided at a multiplicity of infection in a range of about 10 to about25.

[0022] In a preferred embodiment, the bacteriophage-mediated delayedlysis of the strain of E. coli is delayed at higher multiplicities ofinfection relative to lower multiplicities of infection.

[0023] In a preferred embodiment, the biologically active human acidicfibroblast growth factor protein contains 154 amino acids. In a morepreferred embodiment, the human acidic fibroblast growth factor proteinhas the sequence as set forth in SEQ ID NO: 8.

[0024] In a preferred embodiment, the promoter is a T7 polymerasepromoter and the E. coli strain is capable of expressing the gene for T7RNA polymerase. In a more preferred embodiment, the gene for T7 RNApolymerase gene is under the control of an inducible promoter. In aneven more preferred embodiment, the inducible promoter is a lac UV 5promoter.

[0025] In an alternate embodiment, the biologically active human acidicfibroblast growth factor protein contains 146 amino acids.

[0026] In another embodiment, the biologically active human acidicfibroblast growth factor protein contains 140 amino acids.

[0027] In another embodiment of the invention, the biologically activehuman acidic fibroblast growth factor protein contains 134 amino acids.

[0028] In a preferred embodiment, a method of producing a biologicallyactive human acidic fibroblast growth factor protein is provided whichcomprises:

[0029] a) growing a first strain of E. coli cells, which harbor a strainof bacteriophage λ, wherein the bacteriophage λ has atemperature-sensitive mutation,

[0030] b) adjusting the temperature to provide for lysis of the firststrain of E. coli cells and release of the bacteriophage λ,

[0031] c) providing a second strain of E. coli cells which have beentransformed with a plasmid having at least one copy of an expressiblegene encoding said biologically active human acidic fibroblast growthfactor protein, said expressible gene being operably linked to a T7polymerase promoter under the control of an inducible promoter, whereinthe second strain of E. coli cells may be induced to express the genefor T7 RNA polymerase by addition of an inducer;

[0032] d) infecting the second strain of E.coli cells with thebacteriophage λ released from the first strain of E. coli cells; and

[0033] e) incubating the infected second strain of E. coli cells in aculture medium containing the inducer, such that protein is produced andreleased into the culture medium upon lysis of the second strain of E.coli cells, wherein said protein is produced as a soluble,biologically-active protein at a concentration greater than 100microgram/ml.

[0034] Another aspect of the invention encompasses a chemicallysynthesized nucleic acid having the sequence set forth in SEQ ID NO: 1.

[0035] For purposes of summarizing the invention and the advantagesachieved over the prior art, certain objects and advantages of theinvention have been described above. Of course, it is to be understoodthat not necessarily all such objects or advantages may be achieved inaccordance with any particular embodiment of the invention. Thus, forexample, those skilled in the art will recognize that the invention maybe embodied or carried out in a manner that achieves or optimizes oneadvantage or group of advantages as taught herein without necessarilyachieving other objects or advantages as may be taught or suggestedherein.

[0036] Further aspects, features and advantages of this invention willbecome apparent from the detailed description of the preferredembodiments which follow.

BRIEF DESCRIPTION OF THE DRAWINGS

[0037] These and other feature of this invention will now be describedwith reference to the drawings of preferred embodiments which areintended to illustrate and not to limit the invention.

[0038]FIG. 1 shows the chemically synthesized nucleotide sequence forhuman acidic fibroblast growth factor (155 amino acids) (SEQ ID NO: 1)which has been modified by substitution of naturally occurring codonswith codons found in highly expressed E. coli proteins and thetranslated amino acid sequence (SEQ ID NO: 2).

[0039]FIG. 2 shows the modifications made in the chemically synthesizedhaFGF 155 codons. FGF fr HUMECGFB is the sequence obtained from GenBank(at NCBI) (SEQ ID NO: 3). HaFGF 155 is the chemically synthesizedsequence of the present invention (SEQ ID NO: 1).

[0040]FIG. 3 shows the pET24-155@rev construct which contains thechemically synthesized haFGF 155 gene (SEQ ID NO: 1).

[0041]FIG. 4 shows purification of the culture medium containingrecombinant haFGF 154 (SEQ ID NO: 8). In the electrophoregram: lane 1,crude media containing recombinant haFGF 154 (225 mg FGF-1/liter); lane2, Heparin-Sepharose column purified recombinant haFGF 154; lane 3,purification of haFGF 154 by HPLC C-18 column. The unlabelled lane atthe far left contains molecular weight markers.

[0042]FIG. 5 shows the pET24-134@rev construct which contains thechemically synthesized haFGF 134 gene (SEQ ID NO: 4).

[0043]FIG. 6 shows the chemically synthesized nucleotide sequence forhuman acidic fibroblast growth factor (134 amino acids) (SEQ ID NO: 4)which has been modified by substitution of naturally occurring codonswith codons found in highly expressed E. coli proteins and thetranslated amino acid sequence (SEQ ID NO: 5).

[0044]FIG. 7 shows the pET24-140@rev construct which contains thechemically synthesized haFGF 140 gene (SEQ ID NO: 6).

[0045]FIG. 8 shows the chemically synthesized nucleotide sequence forhuman acidic fibroblast growth factor (140 amino acids) (SEQ ID NO: 6)which has been modified by substitution of naturally occurring codonswith codons found in highly expressed E. coli proteins and thetranslated amino acid sequence (SEQ ID NO: 7).

[0046]FIG. 9 shows a 12.5% SDS polyacrylamide gel containing proteinsproduced by the phage-dependent method described herein: lane 1:molecular weight standards, 2 μg each standard; lane 2: 40 μl of culturemedia containing the recombinant FGF 134 protein; lane 3: 40 μl ofculture media containing the recombinant FGF 140 protein; lane 4: 40 μlof culture media containing recombinant interferon α2B; lane 5: 40 μl ofculture media containing recombinant FGF 154 protein; lane 6: 40 μl ofculture media containing recombinant human growth hormone; lane 7: 40 μlof culture media containing recombinant methionine aminopeptidase; lane8: 40 μl of culture media containing β-galactosidase of E. coli.

[0047]FIG. 10 shows a 12.5% SDS polyacrylamide gel containingrecombinant proteins purified according to the presently claimedinvention: lane 1: molecular weight standards; lane 2: 5 μg of purifiedFGF 134 protein; lane 3: 5 μg of purified FGF 140 protein; lane 4: 5 μgof purified FGF 146 protein; lane 5: 5 μg of purified interferon α2Bprotein; lane 6: 5 μg of purified FGF 154 protein; lane 7: 5 μg ofpurified methionine amino peptidase protein; and lane 8: molecularweight standards.

[0048]FIG. 11. Chicken embryo CAM blood vessels on the 14^(th) day ofdevelopment after FGF treatment. Formation of chicken egg CAM new bloodvessels on the 4^(th) day after application of the 154 amino acid formof the haFGF protein. Magnification 3×. FIG. 11A shows the effect of 1μgm of the 154 amino acid form of the haFGF protein. The vessels underapplication are mainly small and show radial growth. FIG. 11B shows thecontrol sample.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0049] While the described embodiment represents the preferredembodiment of the present invention, it is to be understood thatmodifications will occur to those skilled in the art without departingfrom the spirit of the invention. The scope of the invention istherefore to be determined solely by the appended claims.

[0050] The haFGF155 gene encodes a protein containing 155 amino acidresidues (SEQ ID NOS: 1 & 2). The first amino acid of the haFGF 155sequence is the initiator methionine residue, which under normalsituations would be removed during protein synthesis resulting in an FGFprotein of 154 amino acids (SEQ ID NO: 8). However, it has only beenpossible to isolate two shorter aFGF forms from tissue samples. The twoisolated forms contain 140 and 134 amino acid residues. The aFGF formcontaining 140 amino acids is considered complete, while the aFGF formcontaining 134 amino acids is considered to be truncated. It has notbeen possible to extract the aFGF form containing 155 or 154 amino acidsfrom tissue samples. It is not known whether the shorter isoforms occuras a normal function of cell processing or as an artefact producedduring the isolation procedure by specific proteases in the process ofaFGF extraction. Western Blot analysis of the protein produced from theisolated DNA recombinant molecules for the three aFGF forms showed highexpression of the 140 and 134 forms and a low expression level of the154 form.

[0051] In a preferred embodiment of the present invention, the gene forhuman acidic fibroblast growth factor encodes the 154 amino acid form ofthe aFGF protein and is chemically synthesized (SEQ ID NO: 1). Thenucleotide sequence of the haFGF 155 gene has been deduced on the basisof the previously described haFGF 155 amino acid sequence (SEQ ID NO:2). The amino acid sequence of the synthesized haFGF155 gene does notdiffer from those previously described such as the translated sequenceof the human aFGF nucleotide sequence of SEQ ID NO: 3 obtained fromGenBank. However, the preferred nucleotide sequence of haFGF genediffers from those previously described. In a preferred embodiment ofthe present invention, the haFGF 155 gene has been chemicallysynthesized using the codons which are most often used by E. coli forintensively synthesized bacterial proteins. Preferred codon usage tablesfor E coli are well known and available. See, for example,http://pshche.uthct.edu/shaun/Sblack/codonuse.html. Chemical synthesisof polynucleotides was carried out using well known methodology (Edge etal. (1983) Nucleic Acids Research 11 (18): 6419-6435).

[0052] Alternatively, other well known forms of the haFGF gene could beused by those skilled in the art in the practice of the presentinvention including isolated DNA from animal tissues encoding otherforms of the haFGF protein known to those skilled in the art includingthe 154, the 146, the 140 and 134 isoforms and any variants,derivatives, analogs or fragments thererof. The human aFGF proteins maybe used in methods to stimulate angiogenesis. Human aFGF produced by thepractice of the claimed invention may also be used in a composition witha suitable pharmaceutical carrier. Such carriers include, but are notlimited to, saline, buffered saline, water, dextrose and combinationsthereof. In a preferred embodiment, a fibringlue such as Tissucal™(Baxter International, Duarte, Calif.) is used as carrier.

[0053]FIG. 1 shows the complete nucleotide sequence of the haFGF 155gene, as synthesized by the present inventors (SEQ ID NO: 1). A sequencefor human acidic fibroblast growth factor from GenBank (SEQ ID NO:3) wascompared to the chemically synthesized sequence of FIG. 1. Thecomparison is shown in FIG. 2. There are distinctions in 80 codons.

[0054] Expression and cloning vectors typically contain a promoter thatis recognized by the host organism and is operably linked to the haFGFnucleic acid. Promoters are untranslated sequences located upstream (5′)to the start codon of a structural gene (generally within 100-1000 basepairs) that control the transcription and translation of particularnucleic acid sequences, such as the haFGF nucleic acid sequence, towhich they are operably linked. Such promoters typically fall into twoclasses, inducible and constitutive. Inducible promoters are promotersthat initiate increased levels of transcription from DNA under theircontrol in response to some change in culture conditions, e.g., thepresence or absence of a nutrient or a change in temperature. At thistime a large number of promoters recognized by prokaryotic host cellsare known. These promoters are operably linked to haFGF-encoding DNA byremoving the promoter from the source DNA by restriction enzymedigestion and inserting the isolated promoter sequence into the vector.

[0055] Promoters known to those skilled in the art include β-lactamaseand lactose promoter systems (Chang et al. (1978) Nature 275: 615;Goeddel et al. Nature (1979) 281: 544), alkaline phosphatase, and atryptophan (trp) promoter system (Goeddel (1980) Nucleic Acids Research8: 4057; Ep36,776). However, other known bacterial promoters aresuitable. A most preferred promoter is the T7 promoter system. Oneskilled in the art would know how to ligate them to haFGF DNA usingsuitable linkers or adaptors to provide appropriate restriction sites.Promoters may also be used in tandem to achieve higher levels ofexpression.

[0056] Any number of prokaryote host cells are suitable for expressingthe haFGF gene cloned into the vectors described herein. Preferredprokaryotic hosts include eubacteria such as Gram-negative orGram-positive organisms, for example, Enterbacteriaceae such asEscherichia. A most preferred prokaryote host is E. coli.

[0057] Transformation means introducing DNA into an organism so that theDNA is capable of replication, either as an extrachromosomal element orby integration into the chromosome. Transformation of prokaryotic cellsis performed using techniques well known to those skilled in the artsuch as treatment with CaCl₂ or electroporation.

[0058] An important advantage of infecting producer cells with abacteriophage is that the phage causes a profound rearrangement of allmacromolecular synthesis in the bacterial host cells. By turning offtranscription of bacterial genes, phages may increase the copying of thetargeted gene, and consequently, increase the output of desired product.

[0059] In one embodiment of the present super-production system, phage λwith amber-mutations that delay bacterial lysis (e.g., Q⁻ and R⁻mutations) are provided in a strain of E. coli, designated Su°, whichlacks the suppressor responsible for correcting amber-mutations in phageλ. In order to obtain a non-suppressing (Su°) strain of E. coli, Su°clones are selected from the wild-type Su⁺ population. Preferably, aselection marker is inserted into the phage DNA, e.g., tetracycline orampicillin resistance.

[0060] Selection of non-suppressing (Su°) strains of E. coli, forexample, E. coli K 802 was carried out with phage λ cI₈₅₇N_(am7)N_(am53) bla tet (hereinafter λ bla N′). Strain E. coli C600 (λbla N′) served as source of the phage. This phage was obtained byinsertion of plasmid pCV 11 (bla tet) at EcoRI site into single-site(EcoRI) vector carrying ts-mutation in repressor gene (cI₈₅₇). Then twoamber-mutations were introduced into the phage N gene by recombinationin vivo.

[0061] Clones were tested for non-lysogenicity with phage λ clear. Inaddition to phage λ bla N′, phage λ cI₈₅₇ Q_(am117) R_(am54) was used tocheck for suppressor.

[0062] As is known, phage λ N′ mutant is not able to lyse the host cellsand is present in cells in the form of extremely unstable plasmids. Ifthe host cells contain suppressor, the amber-mutation is phenotypicallycorrected, the N protein is synthesized and the phage can developlytically. This difference in the viability of Su+ and Su° cells,infected by λ N′, is used as a basis for selection of spontaneouslyappearing Su° revertants from the E. coli Su⁺ cell population. Phage λwith an inserted plasmid that introduced the ampicillin and tetracyclineresistance markers into cells was used to prevent the nonlysing Su°cells from masking the search for mutants. The phage also containsts-mutation in the repressor gene that permits lytic development of suchphage resulting in cell lysis.

[0063] If the medium supplemented with ampicillin and tetracycline isinoculated with Su⁺ culture after its infection with phage λ bla N′ withsubsequent growth at 43° C., single suppressor-free cells containingphage λ bla N′ in the form of plasmids must develop on plates. Curingthe cells from the phage, we must obtain Su° derivatives of the parentcultures. The method can be subdivided into several stages.

[0064] 1. Infection of Culture With Phage λ bla N′

[0065] The culture E. coli Su+was grown on the M9 medium with maltose at37° C. under intense agitation to a density of 1-2×10⁸ cells/ml. Thecells were infected with phage λ bla N′ at a multiplicity of 5-10particles per cell and incubated for 20 min at 20° C. Under givenconditions, the infection efficiency is about 100%, in addition to thebulk of Su⁺ cells, the phage also infects single Su° cells.

[0066] 2. Selection of Suppressor-Free Cells Containing Marker Phage

[0067] After infection, cells were plated out on agar mediumsupplemented with 12 y/ml tetracycline and 20 y/ml ampicillin and grownat 43° C. In 24 h, single colonies developed, which were replated onagar medium with antibiotics and grown at 37° C.

[0068] 3. Curing of the Selected Clones from Phage λ bla N′

[0069] Since phage λ N′ in the E. coli Su° cells is in the form ofextremely unstable plasmids, in order to cure from the phage theselected clones were plated on selective agar medium without antibioticsand grown at 37° C. The number of cells that had lost the phage in thefirst passage on the medium without antibiotics amounted to 12-35%. Theselection of such cells was carried out by monitoring the loss ofantibiotic resistance and the acquisition of sensitivity to phage λclear.

[0070] 4. Testing of Cells for Repressor

[0071] The ability of phage λ with amber-mutations to form plaques onlawns of cured clones was checked. Isogenic suppressor-free derivativesof the parent E. coli Su⁺ strains are clones, on which phage λ bla N′did not form plaques, phage λ cI₈₅₇ Q_(am117) R_(am54) produced 1-3×10⁵PFU/ml, and phage λ cI₈₅₇ without mutations in genes Q and R produced1×10¹⁰ PFU/ml.

[0072] Using this method, we obtained Su° revertants of E. coli K 802Su⁺. Based on the cell number at the moment of infection and the numberof Su° revertants among them, the frequency of occurrence ofsuppressor-free cells was 3×10⁻⁷.

[0073] In a preferred embodiment, the gene of interest is cloned intopET-24a(+) under the control of the T7 promoter. Preferred genesinclude, but are not limited to, genes encoding human aFGF 134 aminoacid form, human aFGF 140 amino acid form, and human aFGF 146 amino acidform and human aFGF 155 amino acid form. In an alternate embodiment, thegene of interest may be cloned into both a bacterial plasmid and the λphage under the control of appropriate promoters. In a most preferredembodiment, chemically synthesized haFGF 155 gene (SEQ ID NO: 1) iscloned into pET-24a(+) under the control of the T7 promoter.

[0074] The T7 promoter is recognized only by T7 RNA polymerase and isnot recognized by the RNA polymerase of E.coli. The obtained plasmidwith an haFGF gene was transformed into E. coli BL21(DE3). This straincontains the T7 RNA polymerase gene. The T7 RNA polymerase gene is underthe control of the inducible lac Uv5 promoter in order to induce T7 RNApolymerase synthesis only when necessary as this protein is toxic forthe E. coli cell. The induction of the lac promoter is carried out byadding IPTG to the nutrient medium. In order to obtain a haFGF protein,the producer strain, containing the recombinant plasmid with the haFGFgene, is cultured under conditions of intensive aeration to a celldensity of 5×10⁷-5×10⁹ cells in 1 ml at a temperature of 20-40° C. Thenit is infected by lambda phage with the ts-mutation cI repressor genewith a multiplicity from 0.1 to 100 phage bodies per cell and incubationis continued at 20-37° C. for 2-14 hours. Simultaneously with the phage,IPTG at a concentration of 1 mM is introduced.

[0075] Production of the haFGF proteins was achieved by cultivation ofthe producer strain under conditions which slow down the lyticdevelopment of the lambda phage Such conditions include loweredtemperature of cultivation and use of amber mutations in late lambdaphage genes such as Q and R genes.

[0076] The haFGF proteins accumulated in the culture medium as a solubleproteins as a result of the producer strain cells lysis by lambda phage.The output of each haFGF protein generally constituted 20% of thesoluble proteins accumulated in the culture medium. Debris was removedfrom the culture medium by centrifugation. The haFGF can then bepurified from contaminant soluble proteins and polypeptides with thefollowing procedures, which are exemplary of suitable purificationprocedures: by fractionation on an ion-exchange column; ethanolprecipitation, reverse phase HPLC; chromatography on silica;immunoaffinity; SDS-PAGE; ammonium sulfate precipitation; and gelfiltration. In one embodiment, the haFGF recombinant protein waspurified using a C18 HPLC column. In another embodiment, the haFGFrecombinant proteins were applied to heparin sepharose in order toobtain purified haFGF. The purified haFGF was then subjected toautomated amino-terminal sequence analysis for 15 cycles. This analysisindicated that all the initiator methionine at position number 1 ofFGF155 had been removed during synthesis resulting in the production ofan FGF molecule containing 154 amino acids. The amino acids detected incycles 2-14 of the above analysis were identical to positions 2-14 ofFGF155.

[0077] Biological activity of the purified haFGF recombinant proteinswas demonstrated by the ability to generate new vessels (angiogenesis).The assay involved the study of haFGF influence on the formation of newblood vessels using the model of chicken embryonic chorio-allantoicmembrane (CAM).

[0078] A more detailed description of the present invention is providedbelow. While the described embodiment represents the preferredembodiment of the present invention, it is to be understood thatmodifications will occur to those skilled in the art without departingfrom the spirit of the invention. The scope of the invention istherefore to be determined solely by the appended claims.

EXAMPLE 1 Production of Human aFGF 154 by Phage-Dependent Method

[0079] Cultures of Escherichia coli BL21(DE3) (NOVAGEN) were transformedby plasmid pET24-155 @rev (FIG. 3), which contains one copy of thechemically synthesized haFGF 155 gene encoding human acidic fibroblastgrowth factor (155 amino acids) (SEQ ID NO: 1). Cultures of BL21(DE3)contain a single copy of the gene for T7 RNA polymerase under thecontrol of the inducible lac UV5 promoter in the bacterial genome(Studier et al. (1986) J. Mol. Biol. 189: 113-130). Into the plasmidpET-24a(+) (NOVAGEN) was inserted the chemically synthesized haFGF 155gene (SEQ ID NO: 1) under the control of the T7 promoter to produceplasmid pET24-155 @rev. Expression of the haFGF 155 gene begins onlyafter the appearance of T7 polymerase in the cells which is mediatedthrough the induction of the lac UV5 promoter by IPTG.

[0080] Cultures of E. coli BL21(DE3) with pET24-155 @rev were grown withshaking at 37° C. in LB medium, containing 50 μg/ml kanamycin, to adensity of 2×10⁸ cells/ml. Then the cells were infected with phage λcI₈₅₇ Q_(am117) R_(am54) at a multiplicity of about 10 phage bodies per1 bacterial cell and cultivated with shaking at 21° C. for about 14hour. Simultaneously with phage, 1 mM IPTG was introduced into themedium.

[0081] Phage λ cI₈₅₇ Q_(am117) R_(am54) was prepared from lysogeniccultures of E. coli RLMI, which were grown in LB medium at 30° C. withintensive aeration to a density of approximately 1×10⁸ cells/ml. Thelysogenic culture was warmed to 43° C. and incubated for 20 minutes toinactivate cI repressor. The temperature was then decreased to 37° C.and after 60-70 minutes the bacterial cells underwent lysis, with phagesbeing formed at 1-2×10¹⁰ PFU/ml.

[0082] After incubation with the phage-infected cells for 14 hours,debris was removed from the culture medium by centrifugation. Theculture medium, containing the haFGF 154 protein was applied to aheparin sepharose column to obtain pure haFGF 154.

[0083] The culture medium containing the haFGF 154 was analyzed bySDS-polyacrylamide gel electrophoresis under denaturing conditions andstained with Coomassie Blue. An electrophoregram of the culture medium,containing haFGF 154 protein is compared to purified haFGF protein inFIG. 4. Lane 1 shows crude media containing recombinant haFGF 154 (225mg FGF-1/liter). Lane 2 shows Heparin-Sepharose column purifiedrecombinant haFGF 154. Lane 3 shows purification of haFGF 154 by HPLCC-18 column. The unlabelled lane at the far left contains molecularweight markers. The overall purification yield was about 65%.Bioactivity was measured by two different assays, a 3T3 cellproliferation assay and a rat hind limb angiogenesis assay (not shown).The bioactivity was equipotent with FGF-1 obtained from Sigma-Chem. Anassay using chicken embryo chorio-allantoic membrane is shown in Example7, below.

[0084] The production of haFGF 154 protein in phage-infected cultureswas about 20% of the total cellular protein. The molecular weight ofhaFGF 154 was 17,908 Daltons as determined by densitometer Image MasterVDS (data not shown). N-terminal sequence analysis of FGF 154 indicatedan alanine residue at the first position, with no initiator methioninedetected.

EXAMPLE 2 Production of Human aFGF 134 Amino Acid form byPhage-Dependent Method

[0085] Cultures of Escherichia coli BL21(DE3) (NOVAGEN) were transformedby plasmid pET24-134 @rev (FIG. 5), which contains one copy of thechemically synthesized gene encoding human aFGF 134 amino acid form(FIG. 6; SEQ ID NO: 4). The translated amino acid sequence is shown inSEQ ID NO: 5. Cultures of BL21 (DE3) contain a single copy of the genefor T7 RNA polymerase under the control of the inducible lac UV5promoter in the bacterial genome (Studier et al. (1986) J. Mol. Biol.189: 113-130). Into the plasmid pET-24a(+) (NOVAGEN) was inserted thehuman aFGF 134 amino acid form gene under the control of the T7promoter. Expression of the human aFGF 134 amino acid form gene beginsonly after the appearance of T7 polymerase in the cells which ismediated through the induction of the lac UV5 promoter by IPTG.

[0086] Cultures of E. coli BL21(DE3) with pET24-134 @rev were grown withshaking at 37° C. in LB medium, containing 50 μg/ml kanamycin, to adensity of 2×10⁸ cells/ml. Then the cells were infected with phage λcI₈₅₇ Q_(am117) R_(am54) at a multiplicity of about 10 phage bodies per1 bacterial cell and cultivated with shaking at 21° C. for about 14hour. Simultaneously with phage, 1 mM IPTG was introduced into themedium.

[0087] Phage λ cI₈₅₇ Q_(am17) R_(am54) was prepared from lysogeniccultures of E. coli RLMI, which were grown in LB medium at 30° C. withintensive aeration to a density of approximately 1×10⁸ cells/ml. Thelysogenic culture was warmed to 43° C. and incubated for 20 minutes toinactivate cI repressor. The temperature was then decreased to 37° C.and after 60-70 minutes the bacterial cells underwent lysis, with phagesbeing formed at 1-2×10¹⁰ PFU/ml.

[0088] After incubation with the phage-infected cells for 14 hours,debris was removed from the culture medium by centrifugation. Theculture medium containing the haFGF 134 amino acid form was applied to aheparin sepharose column to obtain pure human aFGF 134 amino acid form.

EXAMPLE 3 Production of Human aFGF 140 Amino Acid form byPhage-Dependent Method

[0089] Cultures of Escherichia coli BL21(DE3) (NOVAGEN) were transformedby plasmid pET24-140 @rev (FIG. 7), which contains one copy of thechemically synthesized gene encoding human aFGF 140 amino acid form(FIG. 8; SEQ ID NO: 6). The corresponding protein is shown as SEQ ID NO:7. Cultures of BL21(DE3) contain a single copy of the gene for T7 RNApolymerase under the control of the inducible lac Uv5 promoter in thebacterial genome (Studier et al. (1986) J. Mol. Biol. 189: 113-130).Into the plasmid pET-24a(+) (NOVAGEN) was inserted the human aFGF 140amino acid form gene under the control of the T7 promoter. Expression ofthe human aFGF 140 amino acid form gene begins only after the appearanceof T7 polymerase in the cells which is mediated through the induction ofthe lac UV5 promoter by IPTG.

[0090] Cultures of E. coli BL21(DE3) with pET24-140 @rev were grown withshaking at 37° C. in LB medium, containing 50 μg/ml kanamycin, to adensity of 2×10⁸ cells/ml. Then the cells were infected with phage λcI₈₅₇ Q_(am117) R_(am54) at a multiplicity of about 10 phage bodies per1 bacterial cell and cultivated with shaking at 21° C. for about 14hour. Simultaneously with phage, 1 mM IPTG was introduced into themedium.

[0091] Phage λ cI₈₅₇ Q_(am117) R_(am54) was prepared from lysogeniccultures of E. coli RLMI, which were grown in LB medium at 30° C. withintensive aeration to a density of approximately 1×10⁸ cells/ml. Thelysogenic culture was warmed to 43° C. and incubated for 20 minutes toinactivate cI repressor. The temperature was then decreased to 37° C.and after 60-70 minutes the bacterial cells underwent lysis, with phagesbeing formed at 1-2×10¹⁰ PFU/ml.

[0092] After incubation with the phage-infected cells for 14 hours,debris was removed from the culture medium by centrifugation. Theculture medium containing the haFGF 140 amino acid form was applied to aheparin sepharose column to obtain pure human aFGF 140 amino acid form.

[0093] Human aFGF 140 produced by the method disclosed above hadbiological activity based upon the chick membrane assay (Example 6).

EXAMPLE 4 Production of Human aFGF 146 Amino Acid Form byPhage-Dependent Method

[0094] Cultures of Escherichia coli BL21(DE3) (NOVAGEN) were transformedby plasmid pET24-146 @rev, which contains one copy of the chemicallysynthesized gene encoding human aFGF 146 amino acid form. Cultures ofBL21(DE3) contain a single copy of the gene for T7 RNA polymerase underthe control of the inducible lac Uv5 promoter in the bacterial genome(Studier et al. (1986) J. Mol. Biol. 189: 113-130). Into the plasmidpET-24a(+) (NOVAGEN) was inserted the human aFGF 146 amino acid formgene under the control of the T7 promoter. Expression of the human aFGF146 amino acid form gene begins only after the appearance of T7polymerase in the cells which is mediated through the induction of thelac UW5 promoter by IPTG.

[0095] Cultures of E. coli BL21(DE3) with pET24-146 @rev were grown withshaking at 37° C. in LB medium, containing 50 μg/ml kanamycin, to adensity of 2×10⁸ cells/ml. Then the cells were infected with phage λcI₈₅₇ Q_(am117) R_(am54) at a multiplicity of about 10 phage bodies per1 bacterial cell and cultivated with shaking at 21° C. for about 14hour. Simultaneously with phage, 1 mM IPTG was introduced into themedium.

[0096] Phage λ cI₈₅₇ Q_(am117) R_(am54) was prepared from lysogeniccultures of E. coli RLMI, which were grown in LB medium at 30° C. withintensive aeration to a density of approximately 1×10⁸ cells/ml. Thelysogenic culture was warmed to 43° C. and incubated for 20 minutes toinactivate cI repressor. The temperature was then decreased to 37° C.and after 60-70 minutes the bacterial cells underwent lysis, with phagesbeing formed at 1-2×10¹⁰ PFU/ml.

[0097] After incubation with the phage-infected cells for 14 hours,debris was removed from the culture medium by centrifugation. Theculture medium, containing the haFGF 146 amino acid protein, was appliedto a heparin sepharose column to obtain pure human aFGF 146 amino acidform.

[0098] Human aFGF 146 produced by the method disclosed above hadbiological activity based upon the chick membrane assay (Example 6).

EXAMPLE 5 Purification of haFGF

[0099] The culture medium containing haFGF is diluted with one volume of0.04M KH₂PO₄ buffer, pH 7.0, and applied to a heparin-sepharose columnequilibrated with 0.02 M KH₂PO₄, pH 7.0. The flow rate is adjusted to 80ml/hour. After application of the culture medium containing the haFGFprotein, the column is washed with 0.02M KH₂PO₄ buffer, pH 7.0. Next,the column is washed with 0.02 M KH₂PO₄ buffer containing 0.6M NaCl, pH7.3. Elution is carried out using 0.02 M KH₂PO₄ buffer with 1.5 M NaCl,pH 7.5. All steps are carried out at 4° C.

EXAMPLE 6 Gel Analysis of Recombinant Proteins Produced by thePhage-Dependent Method

[0100] Culture media containing human aFGF 134 amino acid form, humanaFGF 140 amino acid form, and human aFGF 154 amino acid form wereanalyzed by SDS-polyacrylamide gel electrophoresis under denaturingconditions and stained with Coomassie Blue. An electrophoregram ofculture media, containing human aFGF 134 amino acid form, human aFGF 140amino acid form, human and aFGF 146 amino acid form was compared tomolecular weight standards in FIG. 9. Lane 2 shows 30 μl of the culturemedium containing human aFGF 134 amino acid form. Lane 3 shows 30 μl ofculture media containing the recombinant FGF 140 protein. Lane 5 shows30 μl of culture media containing recombinant FGF 154 protein. Lane 1shows 2 μg of each molecular weight standard (Amersham PharmaciaBiotech). From the top, the molecular weight standards are: 94,000;67,000; 43,000; 30,000; 20,100; and 14,400.

[0101] Quantitation of amounts of human aFGF 134 amino acid form, humanaFGF 140 amino acid form, and human aFGF 154 amino acid form in amixture was accomplished by scanning the stained protein bands on apolyacrylamide gel with densitometer Image Master VDS (PharmaciaBiotech). The production of the recombinant proteins in phage-infectedcultures was about 20% of the total cellular protein.

[0102] An electrophoregram containing purified recombinant human aFGF134, haFGF 140, haFGF 146, and haFGF 154 protein was compared tomolecular weight standards (FIG. 10). Lane 2 shows 5 μg of the purifiedaFGF 134 protein. Lane 3 shows 5 μg of the purified human aFGF 140. Lane4 shows 5 μg of the purified human aFGF 146 amino acid form. Theproduction of human aFGF 146 amino acid form in phage-infected cultureswas about 20% of the total cellular protein. Lane 6 shows 5 μg of haFGF154 protein. Lanes 1 and 8 show 2 μg of each molecular weight standard(Amersham Pharmacia Biotech).

EXAMPLE 7 A Method of Studying FGF Influence on the Formation of NewBlood Vessels in the Chicken Embryo Chorio-Allantoic Membrane (CAM).

[0103] The method of studying angiogenesis on the model of chickenembryos (Thomas et al. (1985) Proc. Natl. Acad. Sci, USA 82: 6409-6413)was adapted to determine the effects of the haFGF 154, 146, and 140recombinant proteins on angiogenesis compared to pure brain-derivedacidic fibroblast growth factor. Pure brain-derived acidic fibroblastgrowth factor is a potent angiogenic vascular endothelial cell mitogenwith sequence homology to interleukin.

[0104] The shells of three-day old chicken embryos were sterilized withethyl alcohol. The shell and under shell cover were removed from the airchamber using forceps and the eggs were covered by the bottom of aplastic 35 mm Petri dish. The embryos were incubated at 37° C. for 5-6days. At the end of this period, the embryos were examined and the eggswith well-developed blood vessels of CAM were selected forexperimentation.

[0105] Filter paper disks with deposited gel containing FGF were laid onthe eggs CAM with the gel towards the blood vessels and incubated in athermostat at 37° C. for another 3 days. The gel was prepared in thefollowing way: the tested quantity of FGF was dissolved in 30 μl ofEagle's medium (solution 1); then in 30 μl of Eagle's medium, 10 μg ofheparin was dissolved and 2% of agarose added (solution 2). Then equalvolumes of solution 1 and 2 were mixed and the obtained mixture wasdeposited in aliquots by 60 μl on 12 mm diameter filter paper disks.

[0106] On the 4^(th) day, the filter paper disks were removed. Rich cowmilk (10% milkfat) was injected under CAM in a quantity of about 1 ml orless. The result was a white background against which the CAM vesselswere easily observed.

[0107] The results of the experiment were recorded with a video camerain conjunction with a computer. The formation of new CAM vessel underthe affect of FGF was evaluated by the following parameters: the natureand direction of vessel growth, their quantity and quality (large,medium, small), the presence or absence of anastomosis, etc. These datawere compared with the control samples which had not been exposed toFGF. FIG. 11 shows Chicken embryo blood vessels on the 14^(th) day ofdevelopment after treatment with FGF154 produced by the phage-dependentrecombinant method described herein and purified on heparin sepharose asdescribed.

[0108]FIG. 11A demonstrates the correlation between application ofrecombinant FGF154 protein and the formation of new blood vessels. Onthe fourth day after application of 1 μg of FGF154, vessels are mainlysmall and show radial growth (FIG. 11A). Increasing the amount of FGF154to 3 μg results in a corresponding increase in the size of the bloodvessels (not shown). Medium vessels are observed with radial growth. Afurther increase to 4 μg of FGF154 applied (not shown) results indevelopment of large, medium and small blood vessels at 4 days afterapplication. Untreated control is shown in FIG. 11B.

[0109] It will be understood by those of skill in the art that numerousand various modifications can be made without departing from the spiritof the present invention. Therefore, it should be clearly understoodthat the forms of the present invention are illustrative only and arenot intended to limit the scope of the present invention.

1 8 1 630 DNA Artificial Sequence Chemically synthesized sequence forhuman acidic Fibroblast Growth Factor (155 amino acids) using preferredcodons for E. coli 1 gcgtagagga tcgagatctc gatcccgcga aattaatacgactcactata ggggaattgt 60 gagcggataa caattcccct ctagaaataa ttttgtttaactttaagaag gagatataca 120 t atg gct gaa ggg gaa atc acc acc ttt aca gcgtta acg gag aaa ttt 169 Met Ala Glu Gly Glu Ile Thr Thr Phe Thr Ala LeuThr Glu Lys Phe 1 5 10 15 aac ctt ccg ccc ggg aat tac aaa aaa ccc aagctt ctt tac tgc agt 217 Asn Leu Pro Pro Gly Asn Tyr Lys Lys Pro Lys LeuLeu Tyr Cys Ser 20 25 30 aac gga gga cac ttc ctg cga att ctg cca gat ggcaca gta gat ggg 265 Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly ThrVal Asp Gly 35 40 45 act cgc gat cgc tcc gac cag cac att cag ctg caa ctctcg gcc gaa 313 Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln Leu SerAla Glu 50 55 60 agc gtt gga gag gtc tat atc aag tcg acg gag act ggc cagtac ctt 361 Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln TyrLeu 65 70 75 80 gcc atg gac acc gat ggg ctt ctg tat ggc tca cag acg cctaac gaa 409 Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro AsnGlu 85 90 95 gaa tgc ttg ttt cta gaa aga cta gaa gaa aac cat tac aac acgtac 457 Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr Tyr100 105 110 ata tcg aaa aaa cat gca gag aag aac tgg ttt gta ggc ctt aaaaaa 505 Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys Lys115 120 125 aat ggt tcc tgt aag cgt gga cca cgg act cac tat ggc caa aaggct 553 Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr His Tyr Gly Gln Lys Ala130 135 140 atc ttg ttc ctg cca cta cca gtg agc tcc gac taa g gatccgaatt600 Ile Leu Phe Leu Pro Leu Pro Val Ser Ser Asp * 145 150 155 cgagctccgtcgacaagctt gcggccgcac 630 2 155 PRT Homo sapiens 2 Met Ala Glu Gly GluIle Thr Thr Phe Thr Ala Leu Thr Glu Lys Phe 1 5 10 15 Asn Leu Pro ProGly Asn Tyr Lys Lys Pro Lys Leu Leu Tyr Cys Ser 20 25 30 Asn Gly Gly HisPhe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp Gly 35 40 45 Thr Arg Asp ArgSer Asp Gln His Ile Gln Leu Gln Leu Ser Ala Glu 50 55 60 Ser Val Gly GluVal Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr Leu 65 70 75 80 Ala Met AspThr Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn Glu 85 90 95 Glu Cys LeuPhe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr Tyr 100 105 110 Ile SerLys Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys Lys 115 120 125 AsnGly Ser Cys Lys Arg Gly Pro Arg Thr His Tyr Gly Gln Lys Ala 130 135 140Ile Leu Phe Leu Pro Leu Pro Val Ser Ser Asp 145 150 155 3 468 DNA Homosapiens 3 atggctgaag gggaaatcac caccttcaca gccctgaccg agaagtttaatctgcctcca 60 gggaattaca agaagcccaa actcctctac tgtagcaacg ggggccacttcctgaggatc 120 cttccggatg gcacagtgga tgggacaagg gacaggagcg accagcacattcagctgcag 180 ctcagtgcgg aaagcgtggg ggaggtgtat ataaagagta ccgagactggccagtacttg 240 gccatggaca ccgacgggct tttatacggc tcacagacac caaatgaggaatgtttgttc 300 ctggaaaggc tggaggagaa ccattacaac acctatatat ccaagaagcatgcagagaag 360 aattggtttg ttggcctcaa gaagaatggg agctgcaaac gcggtcctcggactcactat 420 ggccagaaag caatcttgtt tctccccctg ccagtctctt ctgattaa 4684 630 DNA Artificial Sequence Chemically synthesized sequence for humanacidic Fibroblast Growth Factor (134 amino acids) using preferred codonsfor E. coli 4 gcgtagagga tcgagatctc gatcccgcga aattaatacg actcactataggggaattgt 60 gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaaggagatataca 120 t atg aat tac aaa aaa ccc aag ctt ctt tac tgc agt aac ggagga cac 169 Met Asn Tyr Lys Lys Pro Lys Leu Leu Tyr Cys Ser Asn Gly GlyHis 1 5 10 15 ttc ctg cga att ctg cca gat ggc aca gta gat ggg act cgcgat cgc 217 Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp Gly Thr Arg AspArg 20 25 30 tcc gac cag cac att cag ctg caa ctc tcg gcc gaa agc gtt ggagag 265 Ser Asp Gln His Ile Gln Leu Gln Leu Ser Ala Glu Ser Val Gly Glu35 40 45 gtc tat atc aag tcg acg gag act ggc cag tac ctt gcc atg gac acc313 Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr Leu Ala Met Asp Thr 5055 60 gat ggg ctt ctg tat ggc tca cag acg cct aac gaa gaa tgc ttg ttt361 Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn Glu Glu Cys Leu Phe 6570 75 80 cta gaa aga cta gaa gaa aac cat tac aac acg tac ata tcg aaa aaa409 Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr Tyr Ile Ser Lys Lys 8590 95 cat gca gag aag aac tgg ttt gta ggc ctt aaa aaa aat ggt tcc tgt457 His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys Lys Asn Gly Ser Cys 100105 110 aag cgt gga cca cgg act cac tat ggc caa aag gct atc ttg ttc ctg505 Lys Arg Gly Pro Arg Thr His Tyr Gly Gln Lys Ala Ile Leu Phe Leu 115120 125 cca cta cca gtg agc tcc gac taaggatccg aattcgagct ccgtcgacaa 556Pro Leu Pro Val Ser Ser Asp 130 135 gcttgcggcc gcactcgagc accaccaccaccaccactga gatccggctg ctaacaaagc 616 ccgaaaggaa gctg 630 5 135 PRT Homosapiens 5 Met Asn Tyr Lys Lys Pro Lys Leu Leu Tyr Cys Ser Asn Gly GlyHis 1 5 10 15 Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp Gly Thr ArgAsp Arg 20 25 30 Ser Asp Gln His Ile Gln Leu Gln Leu Ser Ala Glu Ser ValGly Glu 35 40 45 Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr Leu Ala MetAsp Thr 50 55 60 Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn Glu Glu CysLeu Phe 65 70 75 80 Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr Tyr IleSer Lys Lys 85 90 95 His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys Lys AsnGly Ser Cys 100 105 110 Lys Arg Gly Pro Arg Thr His Tyr Gly Gln Lys AlaIle Leu Phe Leu 115 120 125 Pro Leu Pro Val Ser Ser Asp 130 135 6 630DNA Artificial Sequence Chemically synthesized sequence for human acidicFibroblast Growth Factor (140 amino acids) using preferred codons for E.coli 6 gcgtagagga tcgagatctc gatcccgcga aattaatacg actcactata ggggaattgt60 gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag gagatataca 120t atg ttt aac ctt ccg ccc ggg aat tac aaa aaa ccc aag ctt ctt tac 169Met Phe Asn Leu Pro Pro Gly Asn Tyr Lys Lys Pro Lys Leu Leu Tyr 1 5 1015 tgc agt aac gga gga cac ttc ctg cga att ctg cca gat ggc aca gta 217Cys Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val 20 25 30gat ggg act cgc gat cgc tcc gac cag cac att cag ctg caa ctc tcg 265 AspGly Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser 35 40 45 gccgaa agc gtt gga gag gtc tat atc aag tcg acg gag act ggc cag 313 Ala GluSer Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln 50 55 60 tac cttgcc atg gac acc gat ggg ctt ctg tat ggc tca cag acg cct 361 Tyr Leu AlaMet Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro 65 70 75 80 aac gaagaa tgc ttg ttt cta gaa aga cta gaa gaa aac cat tac aac 409 Asn Glu GluCys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn 85 90 95 acg tac atatcg aaa aaa cat gca gag aag aac tgg ttt gta ggc ctt 457 Thr Tyr Ile SerLys Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu 100 105 110 aaa aaa aatggt tcc tgt aag cgt gga cca cgg act cac tat ggc caa 505 Lys Lys Asn GlySer Cys Lys Arg Gly Pro Arg Thr His Tyr Gly Gln 115 120 125 aag gct atcttg ttc ctg cca cta cca gtg agc tcc gac taaggatccg 554 Lys Ala Ile LeuPhe Leu Pro Leu Pro Val Ser Ser Asp 130 135 140 aattcgagct ccgtcgacaagcttgcggcc gcactcgagc accaccacca ccaccactga 614 gatccggctg ctaaca 630 7141 PRT Homo sapiens 7 Met Phe Asn Leu Pro Pro Gly Asn Tyr Lys Lys ProLys Leu Leu Tyr 1 5 10 15 Cys Ser Asn Gly Gly His Phe Leu Arg Ile LeuPro Asp Gly Thr Val 20 25 30 Asp Gly Thr Arg Asp Arg Ser Asp Gln His IleGln Leu Gln Leu Ser 35 40 45 Ala Glu Ser Val Gly Glu Val Tyr Ile Lys SerThr Glu Thr Gly Gln 50 55 60 Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu TyrGly Ser Gln Thr Pro 65 70 75 80 Asn Glu Glu Cys Leu Phe Leu Glu Arg LeuGlu Glu Asn His Tyr Asn 85 90 95 Thr Tyr Ile Ser Lys Lys His Ala Glu LysAsn Trp Phe Val Gly Leu 100 105 110 Lys Lys Asn Gly Ser Cys Lys Arg GlyPro Arg Thr His Tyr Gly Gln 115 120 125 Lys Ala Ile Leu Phe Leu Pro LeuPro Val Ser Ser Asp 130 135 140 8 154 PRT Homo sapiens 8 Ala Glu Gly GluIle Thr Thr Phe Thr Ala Leu Thr Glu Lys Phe Asn 1 5 10 15 Leu Pro ProGly Asn Tyr Lys Lys Pro Lys Leu Leu Tyr Cys Ser Asn 20 25 30 Gly Gly HisPhe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp Gly Thr 35 40 45 Arg Asp ArgSer Asp Gln His Ile Gln Leu Gln Leu Ser Ala Glu Ser 50 55 60 Val Gly GluVal Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr Leu Ala 65 70 75 80 Met AspThr Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn Glu Glu 85 90 95 Cys LeuPhe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr Tyr Ile 100 105 110 SerLys Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys Lys Asn 115 120 125Gly Ser Cys Lys Arg Gly Pro Arg Thr His Tyr Gly Gln Lys Ala Ile 130 135140 Leu Phe Leu Pro Leu Pro Val Ser Ser Asp 145 150

What is claimed is:
 1. A human acidic fibroblast growth factor proteinhaving the sequence as set forth in SEQ ID NO: 8.